Influenza A virus (IAV) remains a major threat to human health because of its ability to continually change its surface proteins and evade adaptive immunity. This project addresses key aspects of the innate immune response that provide initial protection of the lung against novel IAV strains. Understanding these innate immune responses may explain why some people suffer severe illness with IAV infection or why pandemic IAV strains are more pathogenic than seasonal strains. For example, our recent findings indicate that pandemic IAV strains evade neutralization by important innate immune mediators, including the surfactant proteins A and D (SP-A and SP-D) and anti-microbial peptides (AMPs) like LL-37, defensins and histones. We have sought to exploit understanding of the mechanisms of action of innate immune proteins to develop modified forms of these proteins with increased antiviral activity. We have thus far shown that mutated versions of SP-D, and modified synthetic peptide derived from LL-37, gain the ability to inhibit pandemic IAV. In Aim 1, we will determine at a molecular level how SP-D binds to the IAV hemagglutinin (HA) in order to explain how pandemic IAV evades neutralization and also how our novel mutated forms of SP-D can overcome this and neutralize IAV. SP-A differs from SP-D in that it neutralizes the virus by providing a decoy sialic acid ligand for the HA to bind to. In Aim 2, we will determine why pandemic IAV is resistant to SP-A but sensitive to some other inhibitors (e.g., H-ficolin) that have similar mechanisms of antiviral activity. In Aim 3, similar studies will be done regarding the AMPs with emphasis on LL-37, which does not inhibit pandemic IAV, and the LL-37 derived peptide which does inhibit it. Finally, Aim 4 will explore how the different proteins modulate inflammatory responses to IAV to reduce potentially harmful inflammation (SP-D, SP-A or LL-37) or possibly increase it (histones) in vitro and in vivo. Key techniques for the proposal will include mass spectroscopy of viral membrane glycoproteins (e.g., HA), xray crystallography and molecular modeling, reverse genetics to generate modified viral strains, and in vitro and in vivo viral infection assays. Our collaborators are prominent experts in glycoproteomics and protein structure analysis and together we have developed powerful techniques to understand the molecular basis for viral neutralization by innate proteins at a level of precision not previously achieved. These studies should enable us to develop additional viral inhibitors through structurally informed modification of collectins (SP-A and SP-D) or AMPs.